Single-exonuclease nanocircuits reveal the RNA degradation dynamics of PNPase and demonstrate potential for RNA sequencing.

The degradation process of RNA is decisive in guaranteeing high-fidelity translation of genetic information in living organisms. However, visualizing the single-base degradation process in real time and deciphering the degradation mechanism at the single-enzyme level remain formidable challenges. Here, we present a reliable in-situ single-PNPase-molecule dynamic electrical detector based on silicon nanowire field-effect transistors with ultra-high temporal resolution. These devices are capable of realizing real-time and label-free monitoring of RNA analog degradation with single-base resolution, including RNA analog binding, single-nucleotide hydrolysis, and single-base movement. We discover a binding event of the enzyme (near the active site) with the nucleoside, offering a further understanding of the RNA degradation mechanism. Relying on systematic analyses of independent reads, approximately 80% accuracy in RNA nucleoside sequencing is achieved in a single testing process. This proof-of-concept sets up a Complementary Metal Oxide Semiconductor (CMOS)-compatible playground for the development of high-throughput detection technologies toward mechanistic exploration and single-molecule sequencing.

Effect of MicroRNA-138 on Tumor Necrosis Factor-Alpha-Induced Suppression of Osteogenic Differentiation of Dental Pulp Stem Cells and Underlying Mechanism.

High doses of tumor necrosis factor-α (TNF-α) suppress osteogenic differentiation of human dental pulp stem cells (hDPSCs). In the present study, we aimed to explore the role and potential regulatory mechanism of microRNA-138 (miR-138) in the osteogenic differentiation of hDPSCs after treatment with a high dose of TNF-α. The hDPSCs were cultured in osteogenic medium with or without 50 ng/ml TNF-α. The miR-138 levels were upregulated during osteogenic differentiation of the hDPSCs following TNF-α treatment. The miR-138 overexpression accelerated but miR-138 knockdown alleviated the TNF-α-induced suppression of the alkaline phosphatase activity, calcium deposition, and protein abundance of dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein, and osteopontin during osteogenic differentiation induction of hDPSCs. Additionally, miR-138 overexpression accelerated but miR-138 knockdown alleviated the suppression of the focal adhesion kinase- (FAK-) extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway during osteogenic differentiation induction of hDPSCs under TNF-α treatment. In conclusion, miR-138 accelerates TNF-α-induced suppression of osteogenic differentiation of hDPSCs. Inactivation of the FAK-ERK1/2 signaling pathway may be one of the mechanisms underlying the effect of miR-138. Inhibition of miR-138 expression may be a strategy to weaken the inhibitory effect of high-dose TNF-α on the osteogenic differentiation of hDPSCs.

Abnormal tapetum development in hermaphrodites of an androdioecious tree, Tapiscia sinensis.

Tapiscia sinensis Oliv. (Tapisciaceae) has been proven to be a functional androdioecious species with both male and hermaphroditic individuals, and the pollen viability of males is far higher than that of hermaphrodites. To better understand the causes of the low pollen viability in hermaphroditic flowers, different stages of anther development were observed. We found that hermaphroditic flowers exhibit abnormal tapetum development, resulting in low pollen viability. To clarify the underlying molecular mechanism of abnormal tapetum development in hermaphrodites, quantitative real-time PCR analyses were performed. The results revealed that the expression levels of an important transcription factor for tapetum development and function, T. sinensis DYSFUNCTIONAL TAPETUM1 (TsDYT1), and its potential downstream regulatory genes T. sinensis DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1 (TsTDF1), T. sinensis ABORTED MICROSPORE (TsAMS) and T. sinensis MALE STERILITY 1 (TsMS1) were all significantly downregulated in hermaphrodites compared with males at some key stages of anther development. The amino acid sequence similarity, expression pattern, gene structure and subcellular localization of these genes were analyzed, and the results indicated functional conservation between T. sinensis and homologues in Arabidopsis thaliana. Next, rapid amplification of cDNA end and thermal asymmetric interlaced PCR were employed to clone the full-length cDNA and promoter sequences of these genes, respectively. In addition, results of yeast two-hybrid analysis showed that TsDYT1 can form heterodimers with TsAMS, and yeast one-hybrid analysis demonstrated that TsDYT1 directly binds to the promoter regions of TsTDF1 and TsMS1. TsTDF1 can directly regulate expression of TsAMS, suggesting that a functionally conserved pathway exists between A. thaliana and T. sinensis to regulate tapetum development. In conclusion, the results suggest that abnormal expression of core transcription factors for tapetum development, including TsDYT1, TsTDF1, TsAMS and TsMS1, plays an important role in the abnormal development of the tapetum in T. sinensis hermaphrodites. Furthermore, a hermaphroditic tapetum with abnormal function causes the low pollen viability of hermaphroditic trees. Our results provide new insight into our understanding of the underlying mechanism of why pollen viability is much higher in males than hermaphrodites of the androdioecious tree T. sinensis.

Sanbai Melon Seed Oil Exerts Its Protective Effects in a Diabetes Mellitus Model via the Akt/GSK-3ß/Nrf2 Pathway.

Traditional Chinese medicine (TCM) plays an important role in the treatment of type 2 diabetes mellitus (T2DM). However, the lack of adequate and scientifically rigorous evidence has limited its application in this disorder. Sanbai melon seed oil (SMSO) is used in folk medicine to treat DM; however, only few literature reports exist regarding its mechanism. Herein, we aimed to confirm the antidiabetic activity of SMSO in a T2DM model and further elucidate its possible mechanisms. The T2DM rat model was induced by high-fat and sugar diet and streptozocin (STZ, 40 mg/kg). SMSO was administered at doses of 0.7 g/kg, 1.4 g/kg, and 2.8 g/kg. Several biochemical parameters and antioxidant protein levels were measured to evaluate the hyperglycemic and antioxidant activities of SMSO. Western blotting was performed to determine its potential mechanism. Based on the results, SMSO treatment significantly reduced blood glucose levels, increased plasma insulin, and repaired islet tissue injury in diabetic rats (P < 0.05). To add, it markedly reduced MDA levels and increased that of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Western blot results showed that SMSO induced n-Nrf2 and HO-1 expression and Akt and GSK-3ß phosphorylation in a dose-dependent manner. Further studies showed that LY294002, aPI3K inhibitor, abolished the effects of SMSO on GSK-3ß phosphorylation and Nrf2 nuclear translocation as well as the protective effects on pancreatic ß cells. Together, these results suggest that SMSO regulates the Akt/GSK-3ß/Nrf2 pathway and induces the expression of antioxidant proteins to impede oxidative stress in rats with T2DM.

[Trace Amounts of Phosphorus Removal Based on the in-suit Oxidation Products of Iron or Manganese in a Biofilter].

In order to remove trace amounts of phosphorus from water bodies, a lab-scale biofilter was constructed to investigate the capacity of in situ oxidation products of iron or manganese for phosphorus adsorption. SEM, EDS, BET, and zeta technologies were employed to reveal the adsorption mechanisms. The results indicated that phosphorus could be removed by the oxide products generated from the iron or manganese removal process, at 106.28 µg·mg-1 and 77.98 µg·mg-1, respectively, as shown by the linear relationships between phosphorus removal and the two oxides. SEM, EDS, and BET analysis demonstrated that the BET specific surface areas for the iron- and manganese-rich oxides were 96 m2·g-1 and 67 m2·g-1, respectively, with the former accumulated between the pore spaces of the filtering sand and easily washed out of the layer by backwashing, whereas the latter coated the surface of the filtering sand. Thus, backwashing was favorable for phosphorus adsorption in the iron oxidation process to avoid overaccumulation. Moreover, the zero point of charge of the two oxides indicated electrostatic attraction may have occurred between iron-rich oxide and phosphorus; however, inner-sphere complex reactions obviously occurred for the two oxides because the zero point of charge after phosphorus adsorption decreased to a lower level. In addition, other anions were negatively complexed with the phosphorus on the surface of the oxides, it demonstrated that phosphorus adsorption on the surface of the two oxides seemed to be a specific adsorption.

Direct Measurement of Single-Molecule Adenosine Triphosphatase Hydrolysis Dynamics.

F1-ATPase (F1) is a bidirectional molecular motor that hydrolyzes nearly all ATPs to fuel the cellular processes. Optical observation of labeled F1 rotation against the α3ß3 hexamer ring revealed the sequential mechanical rotation steps corresponding to ATP binding/ADP release and ATP hydrolysis/Pi release. These substeps originate from the F1 rotation but with heavy load on the γ shaft due to fluorescent labeling and the photophysical limitation of an optical microscope, which hampers better understanding of the intrinsic kinetic behavior of ATP hydrolysis. In this work, we present a method capable of electrically monitoring ATP hydrolysis of a single label-free F1 in real time by using a high-gain silicon nanowire-based field-effect transistor circuit. We reproducibly observe the regular current signal fluctuations with two distinct levels, which are induced by the binding dwell and the catalytic dwell, respectively, in both concentration- and temperature-dependent experiments. In comparison with labeled F1, the hydrolysis rate of nonlabeled F1 used in this study is 1 order of magnitude faster (1.69 × 108 M-1 s-1 at 20 °C), and the differences between two sequential catalytic rates are clearer, demonstrating the ability of nanowire nanocircuits to directly probe the intrinsic dynamic processes of the biological activities with single-molecule/single-event sensitivity. This approach is complementary to traditional optical methods, offering endless opportunities to unravel molecular mechanisms of a variety of dynamic biosystems under realistic physiological conditions.

Programmed cell death: a mechanism for the lysigenous formation of secretory cavities in leaves of Dictamnus dasycarpus.

The formation of secretory cavities in Rutaceae has been the subject of great interest. In this study, cytological events that are involved in the lysigenous formation of the secretory cavities in the leaves of Dictamnus dasycarpus are characterized by an interesting pattern of programmed cell death (PCD). During the developmental process, clusters of cells from a single protoepidermal cell embark on different trajectories and undergo different cell death fates: the cell walls of the secretory cells have characteristics of thinning or complete breakdown, while the sheath cells present a predominantly thick-walled feature. A DAPI assay shows deformed nuclei that are further confirmed to be TUNEL-positive. Gel electrophoresis indicates that DNA cleavage is random and does not result in ladder-like DNA fragmentation. Ultrastructurally, several remarkable features of PCD have been determined, such as misshapen nuclei with condensed chromatin and a significantly diffused membrane, degenerated mitochondria and plastids with disturbed membrane systems, multivesicular bodies, plastolysomes, vacuole disruption and lysis of the center secretory cell. Cytological evidence and Nile red stains exhibit abundant essential oils accumulated in degenerated outer secretory cells after the dissolution of the center secretory cell. In addition, explanations of taxonomic importance and the relationship between PCD and oil droplet accumulation in the secretory cavities are also discussed.

[Dynamic changes of content of salvianolic acid in vegetative organs of Salvia miltiorrhiza].

OBJECTIVE: To determine the content of total salvianolic acid in different vegetative organs of Salvia miltiorrhiza and discover the dynamic change rules of tanshinol, protocatechuic aldehyde, caffeic acid and total salvianolic acid during the whole process of grwth. METHODS: HPLC-ECD was used. The separation was performed on Zorbax SB-C18 (150 mm x 4.6 mm, 5.0 microm) column by gradient elution. The mobile phase consisted of CH3OH-0.4% aqueous phosphoric acid. The flow rate was 1.0 mL/min. The detection was done at 0.7 V and the column temperature was 30 degrees C. RESULTS: The highest content of total salvianolic acid in leaf was in June and gradually dropped off till the lowest in December; The content of total salvianolic acid in leaf gradually decreased along with the growing of the leaf. The content of total salvianolic acid in root was high and consistent from July to September, but gradually dropped off till the lowest in November. CONCLUSION: The leaves of Salvia miltiorrhiza can be harvested in strong growth period to achieve the comprehensive use of the herb.

[Analysis of four components in Fufang Danshen Tablets by high performance liquid chromatography with electrochemical and ultraviolet detections].

A rapid and accurate method for the simultaneous separation and determination of protocatechuic acid, protocatechuic aldehyde, caffeic acid and tanshinone II A in Fufang Danshen Tablets by high performance liquid chromatography has been established. The former three constituents were detected by electrochemical array detector (ECD) and the last one by ultraviolet diode array detector (DAD). The separation was performed on a Zorbax SB-C18 column (150 mm x 4.6 mm i.d., 5.0 microm) by gradient elution with methanol and 0.4% aqueous phosphoric acid as mobile phase at a flow rate of 1.0 mL/min. The detections were done at 0.7 V, 270 nm and 30 degrees C. The results showed that the linear ranges were 0.3 - 9.0 mg/L for protocatechuic acid, 1.1 - 54.0 mg/L for protocatechuic aldehyde, 1.1 - 11.1 mg/L for caffeic acid and 5.2 - 52.0 mg/L for tanshinone II A. The average recoveries (n = 6) were 97.8% with relative standard deviation (RSD) of 2.15% for protocatechuic acid, 98.2% with RSD of 2.07% for protocatechuic aldehyde, 97.6% with RSD of 2.18% for caffeic acid and 97.2% with RSD of 2.07% for tanshinone II A. These four components were separated successfully from each other. The sensitivities of detection of protocatechuic acid, protocatechuic aldehyde and caffeic acid with ECD were 11.0 - 25.3 times greater than those with DAD. The study provides a scientific basis for the quality control of Fufang Danshen Tablets.